Cocaine esterase from Pseudomonas sp. NCIMB 40427 for detection of cocaine

ABSTRACT

A cocaine esterase has been isolated from a strain of the bacteria Pseudomonas maltophilta, The cocaine esterase catalyses the debenzoylation of cocaine, This reaction may be used in the detection of cocaine. The enzyme may be incorporated into sensors for this purpose. The cocaine esterase is preferably obtainable from Pseudomonas sp. NCIMB 40427. It catalyzes the debenzoylation of cocaine, has a molecular weight in the unaggregated form of about 120,000 daltons as determined by gel filtration, has esterase activity specifically at the benzoate ester linkage of cocaine, separates at a major band of Rf about 0.2 on PAGE in its aggregated form, and it is completely inhibited by 1 mM phenylmethylsulphonyl fluoride but ineffectively inhibited by 1 mM eserine, each determined at 30° C. with respect to 2 mM cocaine as substrate.

BACKGROUND OF THE INVENTION

This invention relates to a new enzyme isolated from a microorganism,the microorganism that produces this enzyme, the use of this enzyme incatalysing the degradation of cocaine and a method and apparatus for thedetection of cocaine using this enzyme.

DESCRIPTION OF THE PRIOR ART

There is an urgent need for a better method of detection of cocaine inparticulate form and in body fluids. In relation to particulate cocaine,although many different analytical systems have been proposed, most arebased on large pieces of equipment such as mass spectrometry, andrequire specially trained laboratory technicians. Such systems includethin layer chromatography, gas chromatography and high pressure liquidchromatography (HPLC). In relation to body fluids they requireextraction of the sample to remove interfering compounds. Labelledassays have been used but these also require specialist skills to carryout. Some of the above or other prior methods, e.g. mass spectrometry,also require bulky and expensive equipment.

Portable detecting devices for cocaine have recently been developed.Thus, Einceman et al. (Anal. Chem. (1990) 62 1374-1379) have developed aportable ion mobility spectrometer, but it relies heavily on thevolatility of the drug. A similar drawback exists with the piezoelectriccrystal coated detecting device of Ngeh-Ngwainbi et al. (Blosensors &Biomechanics (1990) 5 13-26). A potentiometric sensor has been developedby K. Vytras et al., (Mikrochlmica Acta (1984) [Wein] III 139-148) butthis is very unspecific as it forms a complex with the tertiary aminegroup present in many illicit drugs.

SUMMARY OF THE INVENTION

We have now found an enzyme which can be used in the detection ofcocaine. The enzyme is a cocaine esterase (hereinafter CE) whichcatalyses the debenzoylation of cocaine that is to say its hydrolysis toproduce ecgonine methyl ester and benzoic acid. This enzyme could thusbe used to detect cocaine, by reacting the enzyme with cocaine anddetecting the occurrence of the enzyme-catalysed reaction. One suchmethod of detection would be conductimetric, as the CE reaction willbring about a change in conductance of the solution. Alternatively, themethod of detection could be potentiometric, the CE reaction bringingabout a change in the pH of the solution. Other methods could includeoptical, color,metric, thermometric or amperometric detection of thereaction products, such methods being well known in the art. Accordinglythe Invention includes sensors, especially of the conductrimetric orpotentiometric type for cocaine esterase. These and similar sensors canbe used as the basis for convenient portable sensors for detectingcocaine in body fluids, luggage, clothing etc. of smugglers, traffickersand cocaine users. Accordingly the invention provides an importantadvance in the fight against and control of use of drugs.

The term "cocaine" used throughout the specification comprises the freebase and salts thereof, unless the context requires a more specificmeaning.

In a first aspect the invention provides the cocaine esterase enzyme.The cocaine esterase enzyme catalyses the debenzoylation of cocaine intoecgonine methyl ester and benzoic acid; thus it attacks the benzoateester linkage of cocaine. There is little or no activity at the methylester linkage. Thus, ability to specifically attack the benzoate esterlinkage of cocaine is a distinctive feature of the cocaine esterase ofthe invention, that is not possessed by commercially availableesterases, e.g. porcine liver esterase, horse serumbutyryl-cholinesterase or other esterases such as microbialatropinesterase.

Another distinctive feature of the CE is that it has a native molecularweight in its unaggregated form of about 120,000 Daltons and whenaggregated of about 420,000 Daltons (both as determined by elution froma gel filtration column calibrated with protein markers). By the term"about" we mean to encompass variations which are usual in thedetermination of high molecular weights by this method and certainly toinclude a variation of up to 10%. This molecular weight is considerablydifferent from that of microbial atropinesterase which has a molecularweight of 30,000 Daltons when denatured and 60,000 Daltons in its nativeform (Rorsch et al., Proc. K. Ned. Akad. Wet. (1971) Set. C 74 132-152).

Example 3 hereinafter describes other features of the CE, but it isexpected that it will be possible to vary some of these by changing theconditions of growth of the microorganism which produces it, or by ahigher degree of purification of the enzyme. Accordingly, it is notpreferred to rely on such characteristics as the catalytic activity orthe thermal stability of CE in the most general definition of theenzyme. Any one, or more of them can be permitted (as the contextpermits) as alternative ways of defining the enzyme, but they are bestseen as one or more preferred, additional characteristics to one or moreof those defined above.

The CE is obtained from a bacterial strain isolated from nature. Thebacterial strain is a strain of Pseudomonas maltophilia hereindesignated "MB11L". A Budapest Treaty patent deposit of this bacteriumhas been made at the National Collections of Industrial and MarineBacteria (NCIMB), 23 St. Machat Drive, Aberdeen, AB2 1RY, Scotland onthe 14th June 1991 under the deposit number NCIMB 40427 and allrestrictions on NCIMB 40427 will be irrevocably removed upon theissuance of a patent. Considerable difficulty lies in the exacttaxonomical classification of some members of Pseudomonas maltophilia asthey could equally be classified as Xanthomonas maltophilia. Pseudomonashas been preferred in this instance but this organism may also beclassified within the genus Xanthomonas. This bacterium, together withmutants and variants producing the CE of the invention, are included inthe present invention. The CE can be produced by culturing such abacterium on a source of carbon and nitrogen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 4 plot the course of important stages in the chromatographicpurification of the CE (see Example 2).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Cocaine esterase can be produced by culturing P. Maltophilia on a sourceof carbon and nitrogen. Preferably the source is cocaine itself. Whengrown on glucose or benzoate the activity of the CE is lower than whengrown on cocaine. Additionally D,L-methionine is preferably added to thegrowth medium as this increases the specific activity of the cocaineesterase produced. In an alternative, an equimolar mixture of cocaineand glucose yields an enzyme of high activity. Cultivation is preferablyaerobic at any usual temperature, e.g. within 20° to 40° C. range,preferably 25° to 35° C. To obtain the enzyme the cells can be disruptedin any conventional way. Preferably a cell-free extract is made. Theenzyme is then recovered from the cells or the extract.

Instead of the precise starting organism deposited, a mutant thereof,e.g. derived by gamma-ray irradiation or use of a chemical mutant,induction by culture on another medium etc. or a transconjugant thereofwith another bacterium or an artificially produced variant can be used.

The enzyme or some modification thereof can also be made by recombinantDNA technology using methods well recognised in that art. These mayentail producing the enzyme in another host organism.

The invention is particularly applicable to the detection of grains ofpowdered cocaine (free base or any of its salts) in luggage, cargo, orabout the person, or for the detection of cocaine or its metabolites inbiological fluids especially in urine and blood.

The enzyme of the present invention is of use primarily in the detectionof cocaine. This may be achieved by the use of cocaine esterase enzymealone or in conjunction with one or more other enzymes that will furtherimprove the detection of cocaine.

The methods of making biosensors that rely on conductimetric,amperometric or potentiometric changes in the test reaction are wellknown in the art. UK Patent Application Publication No. 2,231,332A(NRDC) describes such methods and their use in biosensors, the contentsof which in relation thereto is hereby Incorporated by reference.

Any of such methods may be of use in detecting the occurrence of thecocaine esterase reaction and hence in the detection of cocaine.

The cocaine esterase of the Invention has several other uses. It can beused to "clear" the body of excess cocaine after a cocaine overdose.Similarly the organism MB11L can be used to "clear" industrial waste ofcocaine. The term "clear" is used to imply the removal of cocaine to itsless addictive metabolites. Thus the cocaine esterase enzyme of thepresent invention has a use in therapy. Also the enzyme can be used toseparate cocaine Isomers and under the correct conditions, to formcocaine analogues by the reversal of the debenzoylation reaction(biotransformation).

The following Examples illustrate the invention. "TRITON", "SEPHACEL"and "SEPHACRYL" are Registered Trade Marks.

EXAMPLE 1 Preparation of a Cocaine Esterase from the Bacterial StrainPseudomonas maltophila MB11L Materials and Methods

1. Pseudomonas maltophilia (MB11L) was isolated from samples collectedfrom a natural source by enrichment with cocaine as the sole carbonsource.

MB11L was grown in 750 ml of defined medium consisting of Na₂ HPO₄ (4.33g), KH₂ PO₄ (2.65 g), NH₄ Cl (2.0 g), N(CH₂ CO₂ H)₃ (O.1 g) per liter,containing 4 ml/l of a mineral salt solution as described by Rosenbergerand Elsden (J. Gen. Microbiol. (1960) 22 726-739). Cocaine (90% purew/w) was added aseptically as the sole carbon source (10 mM) to 750 mlof the above sterile medium in a 2 liter Ehrlenmeyer flask and shaken at250 rev/min in a shaking incubator. For bulk preparation of bacteria,750 ml of seed culture was asceptically added to a 10 liter culturevessel, containing 9.5 liter of sterile medium. The bulk cultures wereincubated at 30° C., stirred at 500 rev/min with sterile aeration at 18l/min. The growth medium for the bulk culture was 10 mM of said cocainewith the addition of 40 mg/l D,L-methnonine and 700 μl Antifoam Aemulsion (Sigma), which is a 30% aqueous emulsion of silicone polymers.

Cell-free extracts were prepared from cells grown in the above manner.If cells were to be harvested from a 10 liter bulk culture, priorconcentration of the cell broth to 2-3 liters was performed in a rotaryconcentrator fitted with a 200 sq cm membrane of 0.45 μm pore size. Thecells were then washed with 1 liter of growth medium. These cells orthose obtained directly from a smaller volume culture were then pelletedby spinning at 10,000 g for 15 min at 4° C. in a Sorvall RC-5Ccentrifuge fitted with a GS-3 rotor. These pelleted cells wereresuspended in 2 ml of 50 mM MOPS buffer (pH7), per gram wet cellweight. Cells were disrupted by sonication in an MSE Soniprep (FisonsInstruments, FSA Ltd.) using 18×12 μm burst of 15 seconds, alternatedwith 30 seconds of cooling in melting ice. Cell debris and unbrokencells were removed by centrifugation at 48,000 g for 20 min at 4° C. ina Sorvall RC-5C centrifuge using a SS-34 rotor, to give clarifiedcell-free extract.

2. Chemicals

Cocaine free base was obtained by dissolving the hydrochloride (5 g, 15mmoles) in a minimum volume of distilled water, followed by the dropwiseaddition of 1.5 ml of 10M sodium hydroxide solution. The resulting freebase was extracted into 10 ml diethylether and recovered by evaporation.A typical yield was 80%, melting point 96.4°-98° C.

The resolution and identification of cocaine and its breakdown products,and benzoic acid was determined by HPLC analysis at 218 nm or 275 nm onWaters 600 system. The 0.46×25 cm column contained 5 μm Spherisorb(C-18). The mobile solvent phase was as described by Noggle & Clark (J.Assoc. Off. Anal. Chem. (1982) 65 756-761) and Masoud and Krupski (J.Anal. Toxicol. (1980) 4 305-310). The mobile phase was sparged withhelium at 30 ml/min throughout operation.

3. Buffers

The following buffers were used in the purification and characterisationof cocaine esterase.

Buffer A: 50 mM MOPS, 2% glycerol (v/v), 1 mM β-mercapto-ethanol, pH7.0.

Buffer B: 50 mM MOPS, 0.5% (w/v) cholate, 0.1M NaCl, 2% (v/v) glycerol,1 mM β-mercaptoethanol, pH 7.0.

Buffer C: 50 mM sodium borate, 0.5% (w/v) cholate, 0.6M NaCl, 2% (w/v)glycerol, 1 mM β-mercaptoethanol, pH 9.0.

Buffer D: 10 mM K₂ HPO₄ 0.5% (w/v) cholate, 0.1M NaCl, 2% (v/v)glycerol, 1 mM β-mercaptoethanol, pH 6.8 .

4. Assays

Cocaine Esterase

Cocaine esterase in extracts, column fractions and characterisationexperiments (e.g. pH optimum, Michaelis constant determinations) wasroutinely assayed by shaking incubation (250 rpm, 30° C.) of 2 mMcocaine HCl (made up in 1 ml 50 mM MOPS buffer, pH 7.0) with a knownquantity of sample (10-200 μl, 0-0.01 U) for a fixed length of time(10-30 min). The reaction was stopped by the addition of 10 μl ofconcentrated phosphoric acid and protein pelleted at 13,000 rpm in aminifuge. The amount of benzoic acid produced was determined by HPLCanalysis of 50 μl of the supernatant and comparison to standard samples(0-1 mM) treated identically. Standard curves of peak height plottedagainst benzoic acid concentration were linear over the range 0-1 mM.Controls containing no enzyme solution were run to allow for backgroundhydrolysis. An assay was considered invalid if no cocaine remained atthe end of the assay (seen by HPLC). One unit of esterase activity isdefined as the amount of enzyme required to produce 1 μmol of benzoicacid in 1 min at 30° C.

The presence of esterase in a sample could be detected in a similarfashion using gas chromatography to observe benzoic acid production, butno attempt to quantify the assay was made.

Gas chromatography was used in these Examples, but as previouslydiscussed, is an impractical tool for routine portable measurement ofcocaine or cocaine esterase.

All spectrophotometric assays were performed on Perkin Elmer Lambda 3, 5or 7 Dual Beam Spectrophotometers fitted with chart recorders andconstant temperature cell jackets. Readings were against appropriateblanks.

Protein

Protein was routinely assayed by the Coomassie dye-binding method ofBradford (Anal. Blochem. (1976) 72 248-254) using commercially availablereagent and Bovine Serum Albumin standard (Pierce Ltd.--obtained throughLife Science Labs Ltd., Luton). An aliquot (20 μl) of sample containing0.2-1 mg protein/ml was added to 1 ml of reagent and the reactionallowed to develop for 5 min at room temperature prior to reading theabsorbance at 595 nm against a blank of buffer (20 μl) plus reagent (1ml). Comparison to a standard curve of standard values (0-1 mg/ml)allowed calculation of the protein concentration in the sample.

Gel Filtration Standards

The following enzymes were used as molecular weight markers in gelfiltration experiments: Bovine liver catalase, yeast alcoholdehydrogenase and yeast C300 hexokinase (molecular weights 240,000,150,000, 100,000 Daltons respectively). Assays for their activity wereas described in Bergmeyer ((1986) Methods of Enzymatlc Analysis. 3rdEdition, published by V.C.H. Publishers, Weinhelm, Germany). Cytochromec (molecular weight 13,000 Daltons) was detected by virtue of itsabsorbance at 505 nm.

EXAMPLE 2 Purification of Cocaine Esterase

All steps were carried out at 4° C. unless otherwise stated.

A cell free extract of P. maltophilia MB11L was prepared by sonicatingand clarifying in cholate-free buffer A. Cholate was subsequently addedto a concentration of 0.5% (w/v). Prior to DEAE-Anion ExchangeChromatography, the pH was checked and NaCl added to give a finalconductivity of 13 mS/cm. The final salt concentration typicallyapproached 0.1M, but was carefully controlled to ensure binding to DEAESephacel.

1. DEAE-Sephacel Chromatography

Cell free extract, typically containing 900 mg of solubilised proteinwas loaded on to a 27×2.6 cm DEAE-Sephacel (Pharmacia/LKB BiotechnologyLtd.) column pre-equilibrated in buffer B. Loading, washing and elutionof the column was at 15 ml/sq.cm/h. After washing with buffer B until nofurther elution of protein was detected at 280 nm, the cocaine esterasewas eluted with a combined salt and pH gradient 125 ml each of buffer Bto buffer C followed by a wash of 100 ml buffer C. Eluting protein wascollected as 10 ml fractions which were assayed for cocaine esteraseactivity and protein. (As shown in FIG. 1, the enzyme eluted atapproximately 0.4M NaCl, pH 8.3. In this and each of the followingfigures esterase activity is denoted by filled circles and proteincontent by the dotted line. In FIGS. 1 and 2 the dashed line representsthe elution gradient). Active fractions were pooled and dialysed against2 liter quantities of buffer D. (The DEAE-column was routinely cleanedwith 1M NaCl/1% Triton X-100 (v/v) and 0.1M NaOH prior to storage in0.02% (w/v) azide.

2. Hydroxylapatite Chromatography

Dialysed protein (20-200 mg) was routinely loaded on to a 14×1.6 cmBio-gel HT (Bio-Rad Laboratories Ltd.) hydroxlyapatite column. Thecolumn was pre-equilibrated in buffer D, and the protein materialchecked for equivalent pH (6.8) and conductivity (13 mS/cm) prior toloading. A flow rate of 15 ml/sq.cm/h was maintained throughout loading,washing and elution. After loading, the column was washed until nofurther eluting protein was detected at 280 nm with buffer D. Elution ofthe esterase was with a gradient of 30 ml each of buffer D to buffer E(identical to buffer D except that 300 mM potassium phosphate was used)followed by a wash of 30 ml of buffer E. Fractions (3 ml) were collectedand assayed for cocaine esterase activity. A typical elution profile isshown in FIG. 2. Active fractions were pooled and concentrated againstPEG 4000 (20% w/v) to a final volume of approximately 4 ml. (The columnwas routinely cleaned with 1M NaCl/0.5% (w/v) cholate and stored in0.02% (w/v) azide).

3. AcA 44 Gel Filtration Chromatography

Cocaine esterase from the previous step was concentrated toapproximately 4 ml and loaded on to a 60×1.6 cm column of AcA 44Ultrogel (Life Science Laboratories Ltd.) which had beenpre-equilibrated in buffer B. In the presence of cholate, the cocaineesterase would be expected to elute as a protein with a molecular weightof 110,000 Daltons. Elution with buffer B was performed at a flow rateof 4 ml/sq.cm/h and the elute collected as 2 ml fractions. Assays forcocaine esterase activity were performed. A typical elution profile isshown in FIG. 3. Active fractions were pooled for cholate removal.

4. Removal of cholate

The pooled enzyme was diluted 1:10 with buffer A (to bring the cholateconcentration below the critical micelle concentration) and dialysedagainst 2×10 volumes of the same buffer to remove salt and cholate.Cholate removal was monitored by simply adding 20 μl of sample to 1 mlof Coomassie dye binding protein assay reagent, which containsphosphoric acid as a major component. Under the acidic conditions,cholate-containing samples formed a vivid blue precipitate due to theprecipitation of cholic acid. Post-AcA 44 material treated as describedabove did not produce this precipitate, even after subsequentconcentration, indicating effective removal of cholate.

5. Sephacryl S-300 Gel Filtration Chromatography

Concentrated cholate-free esterase was loaded on to a 75×1.6 cm columnof Sephacryl S-300 (Pharmacia LKB Biotechnology Ltd.) pre-equilibratedin buffer A. Subsequent elutton with buffer A was at a flow rate of 4ml/sq.cm/h. In this cholate-free state the esterase was expected toelute as a protein with molecular weight 410,000 Daltons. Collection andassay of 2 ml fractions typically gave a profile as shown in FIG. 4.

Results

In a solubilised extract of P. maltophilia MB11L grown on cocaine as thecarbon source, cocaine esterase was present at a specific activity of0.16 unit (mg protein)⁻¹. It was purified 22-fold as shown in Table 1below.

EXAMPLE 3 Characterisation of Cocaine Esterase pH Optimum

Purified cocaine esterase material (10 μl, 5 μg protein) was incubatedfor 20 min at 30° C. with 2 mM cocaine in a range of buffers: 50 mM BisTris Propane (pH 6.0, 7.0, 8.0, 9.0 and 10.0), 50 mM MOPS (pH 6.5, 7.0and 7.5) and 50 mM Bicine (pH 7.5, 8.5 and 9.0). After removal ofprotein by precipitation with 10 μl concentrated phosphoric acid andcentrifugation at 13,000 rpm in a microfuge, the benzoic acidconcentration in each incubation was determined by HPLC analysis andcocaine esterase activity determined. In each case a control assay wasperformed containing no enzyme, and the assay calibrated using a 1 mMbenzoate standard. All assays were performed in duplicate.

Cocaine esterase displayed a pH optimum range from 7 to 9. Tertiaryamine buffers appeared to have an inhibitory effect on the esteraseactivity.

Effect of Temperature

Samples of cell-free extract were held at various temperatures for atleast 30 min prior to centrifugation. Assays for cocaine esteraseactivity and protein were compared to original levels. The enzyme wasstable up to 30° C., above which enzyme activity fell off rapidly.

A comparison of the thermal stabilities of CE and acetylesteraseactivities in the purified protein preparation (0.015 mg protein) wasperformed at 47° C. in the presence and absence of cholate. The t_(1/2)of acetyl esterase was 4.5 min in the presence of cholate andapproximately 10 min in its absence. CE had a t_(1/2) of 2.5 min in thepresence of cholate and little instability in its absence.

                                      TABLE 1                                     __________________________________________________________________________              Total                                                                             Total                                                                             Overall                                                                            Total                                                                             Specific                                                                           Overall                                                 volume                                                                            activity                                                                          recovery                                                                           protein                                                                           activity                                                                           purification                                  Purification Step                                                                       (ml)                                                                              (U) %    (mg)                                                                              (U/mg)                                                                             (fold)                                        __________________________________________________________________________    Solubilised extract                                                                     150 163 100  1017                                                                              0.16 1.0                                           DEAE-Sephacel                                                                           120 98  60   141 0.70 4.4                                           Anion exchange                                                                Chromatography                                                                Hydroxylapatite HT                                                                      64  57  35   57  1.0  6.3                                           AcA 44 Ultrogel                                                                         28  46  28   20  2.30 14                                            Gel filtration                                                                S-300 Sephacryl                                                                         23  14  9     4  3.50 22                                            Gel filtration                                                                __________________________________________________________________________

Kinetic Characterisation and Substrate Specificity of Cocaine Esterase

Incubations of purified cocaine esterase (1.25 μg of protein) wereperformed in 1 ml of 50 mM bis-tris-propane buffer, pH 8.0, withsubstrates over the concentration range 0-2 mM (0-1 mM for cocaine) at30° C. for 10 min. Reactions were stopped using concentrated phosphoricacid (10 μl) to precipitate protein, which was removed by centrifugationat 13,000 rpm in a Microfuge. Rates of substrate hydrolysis werecalculated from HPLC analyses of the incubations. Non-enzymic rates ofhydrolysis were calculated from control samples containing no enzyme,and the enzyme assays corrected accordingly. Standard samples of therelevant free acids were used to calibrate each assay. From the resultsit was possible to calculate apparent K_(m) and V_(max) values forcocaine esterase against each substrate. Lineweaver-Burke andEadie-Hofstee plots were used to obtain these values (see Fersht, 1985,Enzyme Structure and Mechanism, 2nd Edition, Publishers: W. H. Freeman &Co., Oxford for methods). The mean values are listed in Table 2.

                  TABLE 2                                                         ______________________________________                                                        Apparent K.sub.m                                                                          Apparent V.sub.max                                SUBSTRATE       (mM)        (U/mg)                                            ______________________________________                                        Cocaine         0.36        29.5                                              Ethyl benzoate  1.89        65.0                                              Ethyl 2-hydroxycenzoate                                                                       1.75        Not determined                                    Ethyl 3-hydroxybenzoate                                                                       No activity No activity                                       Ethyl 4-hydroxybenzoate                                                                       No activity No activity                                       ______________________________________                                    

Thus, Michaelis constants obtained using the purified enzyme show thatcocaine esterase has a greater affinity for cocaine than ethylbenzoates, but hydrolyses cocaine at a slower rate than ethyl benzoates.

Molecular Weight Determination

The molecular weight of the native/solubilised enzyme was determined bythe method of Andrews (Blochem. J. (1964) 91 222-233). For thesolubilised enzyme, measurements were performed on a column of SephacrylS-200 (1.6×75 cm). Purified cocaine esterase (2 mg protein) wassolubilised and mixed with marker proteins and the mixture (total volume2 ml) added to the column. The column was eluted with buffer B at 4ml/cm² /h collecting as 1.3 ml fractions. The elution volume of cocaineesterase corresponded to a molecular weight of 110,000 Daltons. Themolecular weight of the aggregated cocaine esterase was performed in asimilar manner on a Sephacryl S-300 column, but no solubilisation wasperformed and elutton was with buffer A. The elution volume of cocaineesterase corresponded to a molecular weight of 410,000 Daltons.

Molecular weight determination was also performed using SDS-PAGE. Thepurified cocaine esterase sample ran as a distinct major bandcorresponding to a molecular weight of 129,000 Daltons when compared tostandard proteins using the method of Shapiro et al. (Blochem. Biophys.Res. Comms. (1967) 28 815-820). This similar value to that of thesolubilised enzyme implies that any effect of detergent/proteininteractions in determining the molecular weight of the solubilisedenzyme were minimal.

Native PAGE analysis of purified CE showed a major band of Rf 0.2, thegel was sectioned using a scalpel blade, and incubations containingsections of the gel with 2 mM cocaine In buffer were analysed forcocaine esterase activity. HPLC analysis of the incubations confirmedthat the region of the gel corresponding to the Rf 0.2 major band,hydrolysed cocaine at a rate higher than the other regions of the gelsampled.

Inhibition Studies

Samples of purified cocaine esterase (0.01 U) were incubated with arange of possible inhibitors (1 mM) at 30° C. for 10 min prior toassaying for activity against cocaine in the usual manner. The possibleinhibitors tested were phenylmethylsulphonyl fluoride (PMSF), eserine,para-hydroxymercuribenzoate (pHMB), benzoate and ecgonine methyl ester.Cocaine esterase activity was determined in a control sample containingno Inhibitor. The results in Table 3 below show that PMSF causes totalinactivation of cocaine esterase activity, whilst the other possibleactive site inhibitors, eserine and pHMB were relatively ineffective asinhibitors.

                  TABLE 3                                                         ______________________________________                                                         % ACTIVITY RELATIVE TO                                       INHIBITOR        CONTROL                                                      ______________________________________                                        1 mM PMSF        0                                                            1 mM Eserine     95                                                           1 mM pHMB        86                                                           1 mM Benzoate    92                                                           1 mM Ecgonine methyl ester                                                                     82                                                           ______________________________________                                    

Activities are relative to that determined with 2 mM cocaine in theabsence of inhibitor (0.9 U/ml=100% activity).

Activities of Commercial Esterases Against Cocaine

A number of commercially available esterase enzymes were screened fortheir ability to hydrolyse cocaine to benzoic acid and ecgonine methylester by incubation with cocaine (2 mM) under conditions reported asoptimal for their usual activity by the manufacturer. Hydrolysis ofcocaine was monitored by HPLC and the results are listed in Table 4.Control incubations containing no enzyme were performed under each setof conditions to allow accurate assaying of enzyme activity. As none ofthe enzymes tested showed appreciable activity compared to that shownagainst their usual substrate, the cocaine esterase from MB11L, appearsunique in its ability to attack the benzoate ester linkage of cocaine.

                                      TABLE 4                                     __________________________________________________________________________    Screen of esterases for activity against cocaine                              Esterase activity with cocaine was monitored by HPLC as                       described earlier. Reaction mixtures contained 2 mM cocaine in                50 mM buffer of pH suited for maximum enzyme activity in each                 case. The activities of the enzymes against cocaine are compared              to their usual substrates in the table below.                                                          SPECIFIC ACTIVITY                                                 SPECIFIC ACTIVITY                                                                         (U/mg)                                                            (U/mg)      AGAINST USUAL                                        ENZYME       AGAINST COCAINE                                                                           SUBSTRATE                                            __________________________________________________________________________    Porcine pancreatic lipase                                                                  0           50                                                   Orange peel acetylesterase                                                                 0           6.2                                                  Porcine liver esterase                                                                     0.833       20                                                   Horse serum butyryl-                                                                       0.098       14                                                   cholinesterase                                                                α-chymotrypsin                                                                       0.04        40                                                   (with calcium)                                                                Electric eel acetyl-                                                                       0           320                                                  cholinesterase                                                                Cocaine esterase.sup.1                                                                     3.5         --                                                   Atropinesterase.sup.2                                                                      0           1.5.sup.3                                            __________________________________________________________________________     .sup.1 Purified enzyme from MB11L.                                            .sup.2 A sample of crude extract from Psuedomonas putida PMBL1 grown on       atropine as sole carbon source with 0.05 U of atropine hydrolysing            activity (measured by HPLC) was incubated with 2 mM cocaine for 20 min at     30° C. No breakdown of cocaine was seen by HPLC analysis.              .sup.3 This value is from crude extract studies by Stevens ((1969) PhD        Thesis, University of Leiden, Netherlands). Purified atropinesterase has      specific activity of 500-600 U/mg (Hessing, (1983), PhD Thesis, Universit     of Leiden, Netherlands).                                                 

Effect of Growth Substrate on Cocaine Esterase Activity

MB11L cells were grown in 750 ml batch culture on a range of carbonsources (10 mM) in medium B. After 3 sub-inoculations cells wereharvested and crude extracts prepared. Assays for cocaine esterase andprotein were performed and the results expressed relative to levels seenin extracts from the cells grown on cocaine (0.02 μmol/min/mgprotein=100%), are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                                  RELATIVE ENZYME ACTIVITY                                                      GROWTH SUBSTRATE                                                    ENZYME      Benzoate Citrate  Cocaine                                                                              Glucose                                  ______________________________________                                        Cocaine esterase                                                                          20%      0%       100%   20%                                      ______________________________________                                    

These results clearly demonstrate the low levels of cocaine esterasefrom cells grown in the absence of cocaine (cells grown on benzoate orglucose give extracts possessing only 20% of the specific activity ofcells grown on cocaine, whilst citrate grown cells possess no activity),indicating that cocaine esterase is inducible. Inclusion of 40 mg/lD,L-methionine in the growth medium increased the specific activity ofthe cocaine esterase produced by 20%.

We claim:
 1. An isolated cocaine esterase obtainable from Pseudomonas spNCIMB 40427 or a mutant thereof having the following properties:(1) itcatalyses the debenzoylation of cocaine; (2) it has a molecular weightin the unaggregated form of about 120,000 Daltons as determined by gelfiltration; (3) it has esterase activity specifically at the benzoateester linkage of cocaine, (4) it separates at a major band of Rf about0.2 on PAGE, in its aggregated form; and (5) it is completely inhibitedby 1 mM phenylmethylsulphonyl fluoride but ineffectively inhibited by 1mM eserine, each determined at 30° C. with respect to 2 mM cocaine assubstrate.
 2. A process of producing the cocaine esterase of claim 1which comprises culturing Pseudomonas sp NCIMB 40427 or a mutant thereofcapable of producing the cocaine esterase, together with a source ofcarbon and nitrogen, at a temperature of 20° to 40° C., disrupting thecells and recovering the cocaine esterase from the disrupted cells.
 3. Amethod of detecting cocaine in a sample, comprising adding the cocaineesterase of the sample to claim 1 to debenzoylation of cocaine in thesample to produce ecgonine methyl ester and benzoic acid, and detectingthe occurrence of said debenzoylation.
 4. A method according to claim 3wherein benzoate ions of the benzoic acid liberated in thedebenzoylation are detected.
 5. A method according to claim 4 whereinthe benzoate ions are detected conductimetrically.